We used growth assays with natural seston, measurements of P balance and feeding experiments to test mechanisms of Daphnia food limitation in three eutrophic Dutch lakes and six oligotrophic Michigan lakes. Improved growth and increased P-content in response to P supplementation confirmed P limitation of native Daphnia in the Dutch lakes. However, addition of Synechococcus, a readily assimilated cyanobacterium, improved growth beyond that achieved by P addition alone. Despite similar seston concentrations, Daphnia growth in seston from three deep Michigan lakes (mean 0.14/d) was much lower than in seston from three shallow lakes (0.33/d). Daphnia from the deep lakes exhibited weak responses to P and fatty acid additions, intermediate responses to a P-deficient alga and strong responses to Synechococcus. Higher P assimilation efficiency in shallow lakes (mean 43%) than in deep lakes (20%) showed that digestion resistance can account for the growth differences between lakes. Deviations from P homeostasis further suggest that Daphnia were on the edge of P deficiency. Understanding zooplankton food limitation in nature will require simultaneous testing for energy limitation and nutritional deficiencies.